PROJECT SUMMARY/ABSTRACT Age-related arterial dysfunction is the primary risk factor for cardiovascular diseases (CVD). One possible link between aging and arterial dysfunction is the gut microbiome, a strong modulator of host physiology that is influenced by age and that may provide novel mechanistic targets for preventing or reversing arterial dysfunction. Ongoing studies in our laboratory are the first to indicate that age-related adverse changes in the gut microbiome, termed gut dysbiosis, influence the development of arterial dysfunction. One consequence of gut dysbiosis is increased production of the adverse gut-derived metabolite trimethylamine N-oxide (TMAO), which has been linked to the development of atherosclerosis in both rodent models and humans. Our preliminary data show that TMAO also independently induces arterial dysfunction, suggesting TMAO contributes to declines in arterial function that precede the development of clinical CVD. Further, our pilot data suggest that inhibition of TMAO with the recently identified compound 3,3-dimethyl-1- butanol (DMB) may reverse arterial dysfunction in old mice, suggesting that DMB may hold clinical promise for reducing the age-associated increase in risk of CVD. Indeed, DMB is a naturally-occurring phytochemical with no known adverse effects, making it an excellent candidate for translation to humans. The purpose of this R21 application is to investigate the efficacy of DMB for (1) preventing the development of arterial dysfunction in mice when initiated in midlife (i.e., before the onset of dysfunction), and (2) reversing or, at the least, improving already established arterial dysfunction in old mice. In addition, we will use several state-of-the-art mechanistic probes to gain insight into the mechanisms underlying the proposed beneficial effects of DMB (Aim 3). In particular, we will investigate whether DMB preserves/improves arterial function via suppression of superoxide-driven oxidative stress, the predominant mechanistic process that mediates age- related arterial dysfunction. Overall, the proposed research has the potential to identify a novel therapeutic strategy to prevent and treat age-related arterial dysfunction, thereby reducing risk of CVD, and represents a critical step in the translation of DMB to humans for preventing CVD and other age-related diseases and disorders. Use of a mouse model of human aging affords a unique opportunity to assess the long-term preventive effects of DMB. Furthermore, this research will address 2 recently announced strategic directions of NIA: 1) to understand interactions between the microbiome and aging and its contributions to declining health and function, and 2) to identify compounds that hold the promise of increasing healthy lifespan. Finally, the proposed studies will establish a new experimental translational paradigm (model) for manipulating gut microbial pathways to determine their effects on CV and physiological functions that influence human healthspan.